Masters Theses

Date of Award

8-2001

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Gary Stacey

Committee Members

Beth Mullin, Neil Quigley

Abstract

This work focuses on the genetic regulation of the nodulation genes found in the symbiotic bacterium Bradyrhizobium japonicum. This work specifically addresses the question of what regulates the important transcriptional regulator gene nolA. The first part of this work uses nolA-luxCDABE fusions to show nolA induction and that the nolA promoter region can drive the lux operon. The plasmid-encoded fusion in Escherichia coli was used to show that the NolA protein induces the nolA promoter to drive the lux operon. The level of nolA induction over the course of the E. coli growth curve was determined by measuring the amount of light produced by the protein products of the lux operon. Without NolA, there was an initial spike of light production which tapered off as the culture density increased. In the presence of NolA, there was a second peak of light production at an optical density of 0.4 (measured at A600). A Tn5 with a nolA-luxCDBE transcriptional fusion was introduced into the B. japonicum chromosome, but the B. japonicum cells did not provide sufficient amounts of the fatty acid substrate (i.e., myristolate) for light production. The B. japonicum cells gave off light when an aldehyde substrate (decanal) was added to the culture. The need for exogenous substrate limited the usefulness of the noiA-luxCDABE fusion as we had intended to use it to view nolA induction in growing, intact host plant nodules. The second part of this work focuses on the generation of a Rhizobium species NGR234 mutant unable to induce a plasmid-encoded noiA-lacZ fusion in response to chitin or high cell population density. The mutants were generated by mating a plasmid (pJQ15Sp) bearing a Tn5 with antibiotic resistance markers into JNR1 ceils (R. NGR234 with a plasmid-encoded noiA-lacZ fusion, pBGAIac4). The transposase was encoded on pJQ15Sp outside the insertion sequences. Some mutants (JNR7-9) were selected based on their lack of nolA induction in response to higher culture densities, and other mutants (JNR1Sp1-45) were selected based on their lack of noiA induction in response to chitin. The lack of response was confirmed by β-galactosidase activity assays. The plasmids were isolated from the mutants and checked for Tn5 insertions before Southern blots were done to determine the number of Tn5 chromosomal insertions. Southern blot analysis revealed that these mutants were interrupted in the same gene. Since interrupting a single gene removes R. NGR234's ability to induce the nolA promoter in response to chitin or high culture density, it seems likely that the single interrupted gene's product passes both signals to nolA. One mutant, JNR9, was chosen for further analysis and plant nodulation assays. The interrupted gene of JNR9 was cloned into the cosmid vector pHC79 using the Promega Packagene kit (results confirmed by Southern blot). The resulting cosmid (pJNR9A) was found to have multiple copies of pHC79. It underwent subcloning to make the cosmid pD32A, which has only one copy of pHC79. The cosmid pD32A will be sequenced at a later time. R. NGR234, JNR1, and JNR9 were used in 28-day nodulation assays on soybean, cowpea, mungbean, and siratro plants. On soybean, R. NGR234 causes large lumpy growths on the roots. The mutant JNR9 caused twice as many growths as the R. NGR234 and JNR1. On cowpea and mungbean plants, JNR9 did not nodulate as well as the controls. On siratro plants, it did nodulate as well as the wild-type bacteria. The differences in JNR9's nodulation ability in different plant hosts may indicate differences in the importance of NoiA in the different hosts.

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